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Quantification of Diatom Gene Expression in the Sea by Selecting Uniformly Transcribed mRNA as the Basis for Normalization

Identifieur interne : 000B63 ( Main/Merge ); précédent : 000B62; suivant : 000B64

Quantification of Diatom Gene Expression in the Sea by Selecting Uniformly Transcribed mRNA as the Basis for Normalization

Auteurs : Lee-Kuo Kang [République populaire de Chine] ; Feng-Hsiu Tsui [République populaire de Chine] ; Jeng Chang [République populaire de Chine]

Source :

Applied and Environmental Microbiology [ 0099-2240 ] ; 2012.

RBID : PMC:3416636

Abstract

To quantify gene expressions by quantitative reverse transcription-PCR (Q-RT-PCR) in natural diatom assemblages, it is necessary to seek a biomass reference specific to the target species. Two housekeeping genes, TBP (encoding the TATA box-binding protein) and EFL (encoding the translation elongation factor-like protein), were evaluated as candidates for reference genes in Q-RT-PCR assays. Transcript levels of TBP and EFL were relatively stable under various test conditions including growth stages, light-dark cycle phases, and nutrient stresses in Skeletonema costatum and Chaetoceros affinis, and TBP expression was more stable than that of EFL. Next, the sequence diversity of diatom assemblages was evaluated by obtaining 32 EFL and 29 TBP homologous gene fragments from the East China Sea (ECS). Based on sequence alignments, EFL and TBP primer sets were designed for Chaetoceros and Skeletonema groups in the ECS. An evaluation of primer specificity and PCR efficiency indicated that the EFL primer sets performed better. To demonstrate the applicability of EFL primer sets in the ECS, they were employed to measure mRNA levels of the FcpB (fucoxanthin-chlorophyll protein) gene in diatoms. The results correctly revealed prominent diel variations in FcpB expression and confirmed EFL as a good reference gene.

Url:

http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3416636

DOI: 10.1128/AEM.00935-12
PubMed: 22706063
PubMed Central: 3416636

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PMC:3416636

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<front><div type="abstract" xml:lang="en"><p>To quantify gene expressions by quantitative reverse transcription-PCR (Q-RT-PCR) in natural diatom assemblages, it is necessary to seek a biomass reference specific to the target species. Two housekeeping genes, <italic>TBP</italic>
 (encoding the TATA box-binding protein) and <italic>EFL</italic>
 (encoding the translation elongation factor-like protein), were evaluated as candidates for reference genes in Q-RT-PCR assays. Transcript levels of <italic>TBP</italic>
 and <italic>EFL</italic>
 were relatively stable under various test conditions including growth stages, light-dark cycle phases, and nutrient stresses in <named-content content-type="genus-species">Skeletonema costatum</named-content>
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 expression was more stable than that of <italic>EFL</italic>
. Next, the sequence diversity of diatom assemblages was evaluated by obtaining 32 <italic>EFL</italic>
 and 29 <italic>TBP</italic>
 homologous gene fragments from the East China Sea (ECS). Based on sequence alignments, <italic>EFL</italic>
 and <italic>TBP</italic>
 primer sets were designed for <named-content content-type="genus-species">Chaetoceros</named-content>
 and <named-content content-type="genus-species">Skeletonema</named-content>
 groups in the ECS. An evaluation of primer specificity and PCR efficiency indicated that the <italic>EFL</italic>
 primer sets performed better. To demonstrate the applicability of <italic>EFL</italic>
 primer sets in the ECS, they were employed to measure mRNA levels of the FcpB (fucoxanthin-chlorophyll protein) gene in diatoms. The results correctly revealed prominent diel variations in <italic>FcpB</italic>
 expression and confirmed <italic>EFL</italic>
 as a good reference gene.</p>
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Quantification of Diatom Gene Expression in the Sea by Selecting Uniformly Transcribed mRNA as the Basis for Normalization

Quantification of Diatom Gene Expression in the Sea by Selecting Uniformly Transcribed mRNA as the Basis for Normalization

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Abstract

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